EBV is a ubiquitous human herpes virus that was first discovered in association with the African (endemic or e) form of Burkitt's lymphoma (BL). Subsequently the virus was also found associated with nasopharyngeal carcinoma (NPC) and was shown to be the causative agent of infectious mononucleosis (IM). Infection usually occurs during early childhood, generally resulting in a subclinical manifestation, occasionally with mild symptoms. Infection during adolescence or adulthood, however, can give rise to IM characterized by the presence of atypical lymphocytes in the periphery. The bulk of these lymphocytes are T lymphocytes; however, included in their number are a small population of B lymphocytes infected by EBV. The infection of B lymphocytes may also be accomplished in vitro. Such cells become transformed and proliferate indefinitely in culture and have been referred to as "immortalized", "latently infected" or "growth transformed ". As far as is known, all individuals who become infected with EBV remain latently infected for life. This is reflected by the lifelong continuous presence of small numbers of EBV genome-positive transformed B-cells among the circulating peripheral blood lymphocytes and the continuous but periodic shedding of virus in the oropharynx.
In the vast majority of cases EBV infection results in a lymphoproliferative disease that may be temporarily debilitating, but is always benign and self-limiting. In certain immunosuppressed individuals, however, the result can be full-blown malignancy. This occurs in individuals who are immunosuppressed intentionally, particularly children receiving organ transplants who are treated with cyclosporine A, or opportunistically, as in the case with individuals infected with HIV, or genetically, as in the case of affected males carrying the XLP (x-linked proliferative syndrome) gene. In these cases the resulting malignancies derive from the polyclonal proliferation of EBV-infected B-cells. In addition, in such patients uncontrolled epithelial replication of the virus is detectable in lesions of oral hairy leukoplakia. Thus, the immune response plays a central role in the control of EBV infection.
As mentioned above, EBV is a member of the herpesviruses. It possesses the following structural properties:
The EBV genome consists of a linear double stranded DNA molecule (173,000 base pairs). PA1 The virion consists of a core (proteins and DNA), surrounded by an icosahedral capsid, and a membrane envelope enclosing the capsid. The icosahedral capsid is built up of hexameric and pentameric capsomeres. The membrane envelope consists of a protein/lipid brayer membrane with spikes on its outer surface. The space between the capsid shell and the envelope is filled with amorphous protein, called the tegument. PA1 Like all herpesviruses, EBV is capable of establishing a latent life-long infection in its host subsequent to primary infection. This latency represents a perfect balance between EBV and its human host, controlled by the host's immune system.
To date, most biochemical and biological studies have been performed on three prototype strains of EBV, being B95-8 (transforming virus produced in a marmoset cell line), P3HR1 (non-transforming virus produced by a Burkitt's lymphoma tumor cell line) and Raji (latent virus in a Burkitt's lymphoma tumor cell line).
During the last few years the entire DNA sequence of the prototype virus strain, B95-8, has been determined. Analysis of this sequence has resulted in the identification of more than 80 open reading frames (Baer et al., 1984, Nature 310, p. 207-211).
The biology of EBV poses a special problem to investigators because its biological characteristics (latent infection) do not lend itself to the classic virus analysis. Furthermore, its cell and host range are effectively limited to human (and those of a few higher primates) B-lymphocytes and epithelial cells which are generally not amenable to culture in vitro. In addition, the absence of a fully permissive cell type, one in which the virus lytically replicates, has severely limited the ability to produce large amounts of the virus.
DNA molecules of B95-8, P3HR1 and Raji isolates have been the prototypes for detailed restriction endonuclease mapping, for cloning into Escherichia coli (E.coli) plasmids and in bacteriophage lambda, and for nucleotide sequencing.
The EBV genome consists of a single double-stranded DNA molecule built up of unique and tandemly repeated DNA elements. Each end of the DNA molecule contains multiple terminal sequences which permit covalently linking and circularization of the genome. In virus particles the EBV genome is only detectable in a linear form. On the contrary, it exists as a circular episome inside the nucleus of latently infected cells.
The internal repeat sequences, IR1 to IR4, separate the EBV genome into 5 unique regions. The U2 and U3 regions vary extensively among different EBV isolates, the former being almost entirely deleted in the P3HR-1 strain of EBV.
The nomenclature for EBV reading frames is based on their position in the virus genome. The names begin with the initials of the BamH1 or EcoR1 restriction fragment where expression begins. The third letter in the name is L or R, depending or whether the expression is leftward or rightward on the standard map. (So BLLF2 is the second leftward reading frame starting in BamH1 restriction fragment L.) The serological classification of virus antigens in the productive cycle of EBV is based on different fluorescence techniques. Antigens specifically detected by means of the anticomplement immunofluorescence technique in the nucleus of fixed, latently infected B-cells (e.g. Raji-cells) are classified as Epstein-Barr nuclear antigens (EBNA). Upon activation of viral gene expression by chemical or viral factors, a class of early antigens (EA) is detected whose synthesis is not blocked by inhibition of viral DNA synthesis. Dependent on the type of fixative used (methanol or acetone) two distinct sets of EA are detectable, EA.sub.R and EA.sub.D. EA is detectable by indirect immunofluorescence in the cytoplasm and nucleus of induced cells. Following onset of viral DNA synthesis (and depending upon it) virus structural proteins (VCA) are synthesized which are detectable by indirect immunofluorescence in the cytoplasm and nucleus of virus producer cells (e.g. P.sub.3 HR.sub.1 cells). On the surface of viable infected cells, induced for virus production, a set of antigens (MA) is detectable by indirect immunofluorescence. These antigens can also be found on the viral envelope and are important targets for virus neutralization. Detection of EBV-specific antibodies in human sera can routinely be performed by serological techniques as described by Menke and Henle (Human Pathology, 5, 551-565, 1974).
Based upon biochemical and immunofluorescence data it is possible to distinguish five different classes of antigen molecules. The different viral polypeptides are designated by their molecular weight, and no common nomenclature has been established except for the virus envelope proteins.
The five different groups of antigens are:
A. The group of antigens which are expressed during a state of latency (EBNAs and LMPs). PA0 B. The group of antigens which are responsible for genome activation and initial induction of viral replication (IEA). PA0 C. The group of antigens which are induced by IEA gene products and which are required for replication of viral DNA; these antigens are mostly viral enzymes (EA). PA0 D. The group of antigens which are structural components of the viral particle and are expressed late in the viral replication cycle (VCA), after initiation of viral DNA synthesis. PA0 E. The group of antigens which are expressed in the cell membrane of the infected cell (MA).
The viral capsid antigens (VCA) of EBV.
For this antigen complex it is of concern that comparison of EBV specific proteins identified in different studies is difficult because of variations in polyacrylamide gel systems, cell lines and chemical inducers used, and the sera employed. Dolyniuk et al. (1 979) described a total of 33 proteins associated with purified virions. Differential solubilization with detergents suggest that the nucleocapsid is composed of at least seven proteins. An important component of the VCA complex is the major capsid protein (MCP). The EBV-MCP is encoded within the BcLF1 reading frame of the viral genome (Bear et al., 1984) and expressed as a 153-160 kDa non-glycosylated protein in EBV-producer cell lines, with a pl of 7.5 to 9.0. This protein is synthesized in the cytoplasm in a soluble form and then transported to the nucleus, where it condenses into capsids and is no longer solubilized by detergents. Another major VCA component has a molecular weight of 125 kDa and is glycosylated. This protein is encoded within the BALF4 reading frame of the viral genome. Although this glycoprotein was classified originally as a VCA component, recent findings indicate that it might in fact be associated with cytoplasmic and nuclear membrane structures.
Experiments described previously (J. M. Middeldorp and P. Herbrink, J.Virol.Meth., 21, 133-146, 1988) were aimed at the identification and characterization of diagnostically relevant EBV marker proteins in relation to different EBV diseases. This was done by using immunoblot strips containing antigens prepared from the virus producer cell line HH514-C16 (a superinducible derivative of P3HR1), induced for the expression of VCA/EA or EA, and from the EBV negative cell lines Ramos and Bjab. Cell lines which carry the EBV genome in a (fully) latent state, X50-7 and JC-5, can be used to study EBNA/LMP specifically.
Patterns of EBV antibody responses were studied in sera of healthy seropositive blood donors, in sera of IM patients and chronic IM patients or patients with EBV-associated tumors like nasopharyngeal carcinoma. Polyclonal and monoclonal antibodies reactive with defined EBV genome products can be used to characterize some of the protein bands detected in this experimental system. These studies however only described proteins or polypeptides with a certain molecular weight. No information was available as to the coding sequence on the EBV genome for these proteins. Nor was it known whether immunoreactive bands on immunoblots were due to reactivity with single or multiple proteins of the same molecular weight.
With immunoblot techniques it is possible to detect an EBV antigen with a molecular weight of 18 kDa. This protein is not expressed when phosphonoacetic acid (PAA) is used to block viral DNA synthesis and is detected by all sera which contain anti-VCA antibodies, which indicates that it is a VCA-related component. Another VCA component is a protein with a molecular weight of 40 kDa. Many of the viral capsid antigens are associated with the nuclear pellet.
At present EBV specific serodiagnosis is accomplished by rather subjective immunofluorescence tests. Progress to more simple and uniform diagnosis (e.g. ELISA) is hampered because bulk production and purification of viral antigens are not possible using standard virus producing cell lines. The only way to achieve this would be to use alternatively prepared EBV antigen(s). These EBV antigens could be prepared with either genetic engineering techniques or synthetic peptide techniques.